This application is a 371 of PCT/US00/05387, filed Feb. 28, 2000.
This invention relates to methods for detecting epithelial cancer.
In another respect the invention pertains to methods for selectively killing epithelial cancer cells.
In a further aspect, the invention concerns methods for detecting epithelial cancer cells in the presence of normal cells and/or for selectively killing such cells, in which the mitochondria of cancer cells retains a mitochondrial marking agent for a time sufficient to permit identification and/or killing such cells.
As used herein, the following terms have the indicated meanings:
xe2x80x9cCancerxe2x80x9d or xe2x80x9ccancerousxe2x80x9d cells are used in the broad sense, to include invasive cancer cells, cancer-in-situ cells and severely dysplastic cells.
xe2x80x9cMitochondrial marking agentxe2x80x9d means a compound that is selectively taken up by the mitochondria of living cancer cells and is selectively retained in cancer cells for a time sufficient to permit identification and/or killing or incapacitation thereof.
xe2x80x9cKillingxe2x80x9d of cells means either causing cell death or cell changes that render a cell incapable of reproduction and metastasizing.
xe2x80x9cAdductxe2x80x9d means the reaction product of a mitochondrial marking agent and a cancer chemotherapeutic agent.
In-vivo diagnostic procedures for detecting malignant and premalignant epithelial lesions or carcinomas, employing dye compositions that selectively xe2x80x9ccolorxe2x80x9d tissues that are abnormal due to dysplasia, hyperplasia, tumorigenesis and other active surface lesions, are known in the art. These diagnostic methods employ a dye that imparts color to a cancerous substrate, which is then detectable under light at visible wavelengths or a fluorescent dye that imparts color to the substrate, which is then detectable when illuminated by light at wavelengths outside the visible spectrum.
For example, procedures employing fluorescein and fluorescein derivatives are disclosed in Chenz, Chinese Journal of Stomatology (27:44-47 (1992)) and Filurin (Stomatologiia (Russian) 72:44-47 (1993)). These procedures involve application of the dye, followed by examination under ultraviolet light to detect the cancerous/precancerous tissue, which is selectively fluorescent. Another prior art procedure involves rinsing the epithelium with toluidine blue, followed by normal visual examination to detect any selectively stained tissue. Such procedures are disclosed, for example in the patents to Tucci et al. (U.S. Pat. No. 5,372,801) and Mashberg (U.S. Pat. No. 4,321,251). Use of certain other thiazine dyes and oxazine dyes in an analogous manner is disclosed in U.S. Pat. No. 5,882,627 to Pomerantz.
Heretofore, it was theorized that such dyes selectively xe2x80x9cmarkedxe2x80x9d cancerous tissue because it was retained in the relatively larger interstitial spaces between the cells of cancerous tissue and would not efficiently penetrate the normally tight intracellular junctions of normal tissue or be selectively retained in such relatively smaller spaces.
Contrary to the belief that toluidine blue selectively marks cancerous epithelial tissue because it is selectively retained in the relatively larger interstitial spaces between cancer cells, the mechanism of such selective staining of epithelial tissue by cationic dyes, e.g., dyes such as rhodamine, fluoresceins, oxazine and thiazine dyes (including toluidine blue) and other cationic supravital marking agents is the selective uptake and selective retention of the agent in the mitochondria of cancer cells. In turn, this selective staining of and retention in the mitochondria is apparently due to the higher electrical potential (negative charge on the inside of the membrane) of cancerous mitochondrial cells as compared to normal cells. See, e.g., Chen et al., Cancer Cells 1/The Transformed Phenotype, 75-85 (Cold Spring Harbor Laboratory, 1984); Lampidis, et al., Cancer Research 43, 716-720 (1983).
In fact, the selective marking and retention of the mitochondria of cancer cells by supravital cationic dyes and other supravital cationic marking agents are related to one of the very characteristics of cancer cells that appears to be responsible for their rapid cloning growth and metastasizing ability, namely, that the higher electrical potential of the mitochondria of cancer cells is the source of cellular energy and is the driving force for ATP (adenosine triphosphate) product of the cells.
I have now discovered a method for in-vivo detection of cancerous epithelial cells by selective marking of the mitochondria thereof. My method comprises the steps of delivering to tissue in the locus of a suspect cancerous site on the epithelium (which contains both normal and cancerous cells), with a cationic supravital mitochondrial marking agent other than rhodamine, causing said agent to be taken up and selectively retained in the mitochondria of cancer cells. The cancerous cells are then detectable by any suitable method, for example, instrumental or visual examination under visible light or under light of selected invisible wavelengths.
In a further embodiment, after the marking agent is taken up by the mitochondria, a rinse reagent is applied to the locus of the suspect cancerous site, thus enhancing the rate of release of the agent from the mitochondria of the normal cells and further increasing the selectivity of the diagnostic method.
According to another embodiment of the invention, I provide a method for selectively killing cancerous epithelial cells comprising the step of contacting cancerous cells in the locus of a suspect cancerous site with a cationic supravital mitochondrial marking agent, to cause cell death or to render the cancer cells substantially incapable of multiplication. The marking agent can be delivered to the cancer cells in a single discrete dose, or continuously, or in repeated discrete doses, with or without employing a rinse reagent after each dose.
In a further embodiment of the invention, I provide a method of improving the selectivity of cancer chemotherapeutic agents comprising the steps of forming an reaction product of a cationic supravital agent and a chemotherapeutic agent and delivering the reaction product to cancerous epithelial cells.